research paper alveolar macrophage - derived …...derived from macrophages, epithelia and...

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INTRODUCTION

Acute lung injury (ALI) is a common clinical lung

disease, and can cause death, and the survivals may

develop acute respiratory distress syndrome (ARDS)

due to fibrotic repair of the lung. ARDS is a devastating

clinical syndrome characterized by non-cardiogenic

pulmonary edema, respiratory distress and hypoxemia

[1]. Although the ARDS-associated mortality has

decreased in the last decade, still about half of the

patients die, while the survivors suffer from significant

physical and psychological impairments [2–4].

Alveolar macrophages play a demonstrative role during

the pathogenesis of ALI, and the timing and degree of

differentially polarization of macrophages determine the

severity of disease and outcome [5]. Exosomes are

cell-secreted nanosized bi-lipid membrane in the

secretome. Bidirectional communication occurs in micro-

environment via exosomes and microvesicles (MVs) [6].

Exosomes and MVs carry nucleic acids, proteins, and

lipids between different cells of the tumor micro-

environment, which influence a multitude of pathways

biologically. Exosomes are 30–100 nm in diameter and

are generated within larger intracellular multivesicular

bodies [6]. Exosomes and microvesicles (MVs) are

released into the extracellular environment on fusion with

the plasma membrane [6]. MVs generally range from 100

to 1000 nm and are formed when cell components travel

to the plasma membrane to be released by membrane

budding [6]. Actually, the previous studies did not strictly

distinguish exosomes from MVs, from definition to

www.aging-us.com AGING 2020, Vol. 12, No. 7

Research Paper

Alveolar macrophage - derived exosomes modulate severity and outcome of acute lung injury

Cong Ye1, Huiting Li2, Minwei Bao1, Ran Zhuo3, Gening Jiang1, Weixi Wang4 1Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University, Shanghai 200433, China 2Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University, Shanghai 200433, China 3Department of Urology, Zhongshan Hospital, Fudan University, Shanghai 200032, China 4Department of Geriatrics, Zhongshan Hospital, Fudan University, Shanghai 200032, China

Correspondence to: Weixi Wang, Gening Jiang; email: [email protected], [email protected] Keywords: acute lung injury (ALI), macrophages, neutrophils, exosomes, bronchoalveolar lavage fluid (BALF) Received: January 8, 2020 Accepted: February 20, 2020 Published: April 7, 2020

Copyright: Ye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Severe acute lung injury (ALI) can cause death, and the survivals may develop acute respiratory distress syndrome (ARDS) due to fibrotic repair of the lung. Alveolar macrophages play a demonstrative role during the pathogenesis of ALI, and the timing and degree of differentially polarization of macrophages determine the severity of disease and outcome. Exosomes are important mediators of cellular communication and play critical roles during macrophage differentiation, proliferation and function. Nevertheless, the exact effects of alveolar macrophage - derived exosomes on ALI remain unknow. Here, we used lipopolysaccharide (LPS) to induce ALI in mice and analyzed the exosome population in bronchoalveolar lavage fluid (BALF) from macrophages, neutrophils and epithelial cells at different time points after treatment. Our data showed that macrophages were the major secretors for early secreted pro-inflammatory cytokines in the BALF-exosomes, which likely activated neutrophils to produce a variety of pro-inflammatory cytokines and IL-10. IL-10 by neutrophils in BALF-exosomes likely in turn polarized macrophages to M2c, which may be responsible for post-ALI fibrosis. Our study thus reveals a previous non-acknowledged role of BALF-exosomes as a mediator of inflammatory response and cell crosstalk during ALI.

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isolation protocols. Exosomes, the validation of which

includes expression of CD9, CD63, CD81, ALIX and

TSG101, are important mediators for cellular

communication and play critical roles during macrophage

differentiation, proliferation and function [7]. Never-

theless, the exact effects of alveolar macrophage -

derived exosomes on ALI remain unknow.

In this study, we used lipopolysaccharide (LPS) to

induce ALI in mice and analyzed the exosome

population in bronchoalveolar lavage fluid (BALF)

from macrophages, neutrophils and epithelial cells at

different time points after treatment. Our data showed

that macrophages were the major secretors for early

secreted pro-inflammatory cytokines in the BALF-

exosomes, which likely activated neutrophils to produce

a variety of pro-inflammatory cytokines and IL-10.

IL-10 by neutrophils in BALF-exosomes likely in turn

polarized macrophages to M2c, which may be

responsible for post-ALI fibrosis.

RESULTS

Purification of BALF-derived exosomes from ALI-

mice

ALI models were applied to mice and BALF were

isolated at different time points. Exosomes were then

purified from the prepared BALF, confirmed by

enriched expression of CD9, CD63, CD81, ALIX and

TSQ101 in exosomes, compared to the corresponding

total BALF (Figure 1). Hence, exosomes were

successfully isolated from ALI-BALF.

Purification of macrophage-, epithelia- and

neutrophil- derived exosomes from total BALF-

exosomes from ALI-mice

Next, we used precursor cell markers to purify exosomes

derived from macrophages, epithelia and neutrophils,

since these three cell types are the major cell types that

contribute to the cytokine production and secretion after

ALI. The exosomes not from these three cell types could

be derived from T-cells, endothelial cells and other cell

types. The purification strategy included size less than

500nm and being positive for specific precursor cell

markers. Macrophages: positive for both CD11c and

F4/80 (Figure 2A). Epithelia: positive for EpCAM

(Figure 2B). Neutrophils: positive for both CD11b and

Ly6G (Figure 2C). Purified exosomes could be

visualized under electron microscopy (Figure 2D–2F).

Using this methodology, the dynamics of exosome

release from macrophages, epithelial cells and

neutrophils within the alveolar space were determined

during the different phase (1 hour, 4 hours, 8 hours,

16 hours and 32 hours) of LPS-induced ALI in mice.

Release of pro-inflammatory cytokines in BALF-

exosomes by different cells after ALI

First, we examined the release pattern of three key pro-

inflammatory cytokines (TNFα, IL-1β and IL-6) in

BALF-exosomes by different cells after ALI. For

TNFα, we found that it started to increase in

macrophage-derived exosomes as early as 1 hour after

ALI, suggesting that the initial responsive cells to LPS

should be macrophages, and their prompt release of

Figure 1. Purification of BALF-derived exosomes from ALI-mice. RT-qPCR for CD9, CD63, CD81, ALIX and TSQ101 in purified exosomes from BALF after ALI. *p<0.05. N=5.


TNFα suggests that they may be polarized from naïve

macrophages to M1 macrophages (Figure 3A).

Moreover, this early release of TNFα also appeared to

be the peak value by macrophages, since TNFα derived

from macrophages continuously decreased afterward

(Figure 3A). The TNFα by epithelia appeared to

increase from 1 hours after ALI, and likely peaked at 8

hours after ALI, with a relatively slow altering pattern

(Figure 3A). On the other hand, The TNFα by

neutrophils increased from 4 hours after ALI, but

increased dramatically and appeared to be the major

source of TNFα after 8 hours post ALI (Figure 3A).

Together, these data suggest that LPS may first trigger

for M1 polarization of macrophages, which release

TNFα to recruit and differentiate neutrophils to further

produce and secrete TNFα to mediate the early pro-

inflammatory effects after ALI. Similar pattern was

detected for IL-1β (Figure 3B), except for that its

absolute increase, especially the first phase increase by

macrophages, was modest (Figure 3B). The decrease of

IL-1β after peak was also less pronounced (Figure 3B),

and the peak of the modest increase of IL-1β by

epithelia occurred at 4 hours after ALI (Figure 3B). For

IL-6, its prompt release by macrophages did not

Figure 2. Purification of macrophage-, epithelia- and neutrophil- derived exosomes from total BALF-exosomes from ALI-mice. The isolated BALF-exosomes were further purified into macrophage-, epithelia- and neutrophil- derived exosomes, based on differential expression of precursor cell markers. The purification strategy included size less than 500nm and being positive for specific precursor cell markers. (A) Flow chart for macrophage-derived exosomes: positive for both CD11c and F4/80. (B) Flow chart for epithelia: positive for EpCAM. (C) Flow chart for neutrophils: positive for both CD11b and Ly6G. (D–F) EM visualization of exosomes purified from isolated exosomes. Scale bars are 50nm.


decrease after 1 hour, while the contribution to IL-6 by

macrophages and neutrophils was similar after 4 hours

(Figure 3C). Thus, the pro-inflammatory cytokines in

BALF-exosomes after ALI are derived primary from

polarized M1 macrophages at early stage, and from

neutrophils at later stages.

Release of anti-inflammatory cytokines in BALF-

exosomes by different cells after ALI

Next, we examined the release pattern of three key anti-

inflammatory cytokines (IL-4, IL-13 and IL-10) in

BALF-exosomes by different cells after ALI. For IL-4

and IL-13, we detected similar release patterns (Figure

4A, 4B). We found that both IL-4 and IL-13 started to

increase at very late stage after ALI (IL-4 as late as 16

hours, and IL-13 as late as 32 hours) and were

exclusively released by macrophages (Figure 4A, 4B).

These data are consistent with established knowledge

that both IL-4 and IL-13 are produced and secreted by

T-cells and macrophages, but not by other cell types.

The late release of IL-4 and IL-13 by BALF-

macrophages suggests that they may be produced and

secreted by polarized M2 macrophages, possibly in an

autocrine manner to regulate macrophage phenotypic

alternation to obtain appropriate functions for

tissue repairing. While IL-10 was hardly released by

epithelia, the earliest release of IL-10 was detected in

BALF-exosomes derived from neutrophils at 4 hours

after ALI without decrease till 16 hours (Figure 4C). On

Figure 3. Release of pro-inflammatory cytokines in BALF-exosomes by different cells after ALI. (A–C) ELISA for 3 key pro-inflammatory cytokines (TNFα, IL-1β and IL-6) in respective BALF-exosomes at 1 hour, 4 hours, 8 hours, 16 hours and 32 hours after ALI. (A) TNFα. (B) IL-1β. (C) IL-6. *p<0.05. N=5.


the other hand, the earliest release of IL-10 by

macrophages appeared at 16 hours after ALI and further

increased afterwards (Figure 4C). Since IL-10 is a well-

known potent trigger of fibrotic macrophages- M2c,

these data suggest that the differentiation of the fibrotic

macrophage subtype M2c may be initiated by

neutrophils-derived IL-10, and later by IL-10 derived

from differentiated macrophages themselves.

Release of fibrotic cytokines in BALF-exosomes by

different cells after ALI

Finally, we examined the release pattern of two key

fibrotic cytokines (TGFβ and FGF) in BALF-exosomes

by different cells after ALI, since they are related to

outcome of post-ALI tissue repair and ARDS in human.

For both, we detected similar release pattern like IL-10

(late release exclusively by macrophages), which is

consistent with the knowledge that they are mainly M2c

cytokines (Figure 5A, 5B), suggesting that the early

release of IL-10 by neutrophils may be primary

responsible for differentiating M2c, while the late release

of IL-10 by polarized macrophages may be the main

trigger for their production and secretion of TGFβ and

FGF. However, compared to the nearly exclusive release

of TGFβ by macrophages (Figure 5A), FGF was partially

released by epithelia, as early as 4 hours after ALI

(Figure 5B), suggesting that it may be from multisources.

Hence, IL-10 is the initial trigger of fibrosis, and it may

function through activating TGFβ- and FGF-secreting

M2c macrophages. Our findings were then summarized

in a schematic (Figure 6).

Figure 4. Release of anti-inflammatory cytokines in BALF-exosomes by different cells after ALI. (A–C) ELISA for 3 key anti-inflammatory cytokines (IL-4, IL-13 and IL-10) in respective BALF-exosomes at 1 hour, 4 hours, 8 hours, 16 hours and 32 hours after ALI. (A) IL-4. (B) IL-13. (C) IL-10. *p<0.05. N=5.


DISCUSSION

One of the severe outcomes of ALI is ARDS, which is

characterized by chronic inflammation in the airways or

alveoli [8]. The most important cell types that

contribute to the pathogenesis of ALI are macrophages,

epithelial cells and neutrophils, compared to other cell

types including T-cells, endothelial cells and

Figure 5. Release of fibrotic cytokines in BALF-exosomes by different cells after ALI. (A, B) ELISA for 2 key fibrotic cytokines (TGFβ and FGF) in respective BALF-exosomes at 1 hour, 4 hours, 8 hours, 16 hours and 32 hours after ALI. (A) TGFβ. (B) FGF. *p<0.05. N=5.

Figure 6. Schematic of the model. LPS first induces macrophage polarization to M1, which produces and releases TNFα to recruit and activate neutrophils, which further produced and secreted TNFα in an autocrine manner to mediate the early pro-inflammatory responses after ALI. Neutrophils also produce and secrete IL-10, which signals back to macrophages to polarize them to M2c to secrete TGFβ to mediate fibrosis.


mesenchymal cells [9]. It is obvious that the

relationship among these cell types during ALI is

interactive and dynamic, while one cell type “instructs”

another cell type to alter its phenotype, which in turn

talks back. The messages that are transferred among

these cells in the microenvironment (BALF) are

assembled into some small cargos, e.g. exosomes, to be

released from one cell into BALF and then obtained by

another to decipher the signals [10, 11]. Previous

studies have not systemically analyzed the exosomes-

cytokines from different types of cells in the micro-

environment, although it is very important to understand

the crosstalk among different cell types in ALI-

associated immunology.

Here, we used a flow cytometry-based method to address

this question. Co-expression of CD11c and F4/80 was

used to determine macrophage-derived exosomes. CD11c

is an integrin alpha X chain protein, functioning in the

adherence of neutrophils and monocytes to stimulated

endothelium cells, and in the phagocytosis of

complement coated particles. CD11c is a marker for

monocytes and macrophages, but also expressed in

dendritic cells [12, 13]. F4/80 is EGF-like module-

containing mucin-like hormone receptor-like 1 (EMR1),

a member of the adhesion G protein-coupled receptor

family. EMR1 expression in human is restricted to

eosinophils and in mice is nearly restricted to macro-

phages [14]. Thus, co-expressing CD11c and F4/80

faithfully determined the exosomes from macrophage

population. Also, that is the reason that we detected a few

CD11c+F4/80- exosomes, which may be derived from

dentritic cells. On the other hand, we used Epithelial cell

adhesion molecule (EpCAM), a transmembrane glyco-

protein mediating Ca2+-independent homotypic cell-cell

adhesion in epithelia, as a unique marker for determining

epithelia-derived exosomes [15]. Finally, co-expressing

CD11b and Ly6G was used to determine neutrophils-

derived exosomes, since CD11b is expressed by

monocytes/macrophages, neutrophils, granulocytes,

macrophages, and natural killer cells [16], while Ly6G is

nearly exclusively expressed by neutrophils [17]. The

CD11b+Ly6G- population should be mainly macro-

phages-derived exosomes.

The first interesting finding is TNFα, as it is the most

important pro-inflammatory cytokine involved in the early

pathogenesis of ALI. The major sources for TNFα are

macrophages and neutrophils, while macrophages

contribute to most early TNFα, neutrophils secreted most

TNFα after 4 hours. Thus, it may be deducted that the

early TNFα by macrophages should be secreted by the

early macrophages responsive to the ALI-causes, LPS,

and should be the trigger for all later responses. It is well-

known that TNFα is a M1 macrophage cytokine [18], and

thus the release of TNFα from macrophages at 1 hour

after ALI should be from newly polarized macrophages,

M1. The second wave of TNFα came from neutrophils,

and it was clear that these neutrophils were activated later

than those M1 macrophages, and were probably just

activated by them, and even through TNFα [19].

The second important finding is IL-10/TGFβ. IL-10 is a

known stimulator of M2c, the M2 macrophage subtype

characterized with TGFβ production and fibrosis [20].

IL-10 was initially found to be released from neutrophils

during ALI, which was a novel finding, since it was

believed that either macrophages or T-cells may be the

major source of IL-10 [21]. Here, our data showed very

pronounced high IL-10 in the BALF-exosomes derived

from neutrophils, consisting with some previous reports

[22, 23], which suggests that neutrophils may be a major

regulator for the differentiation of macrophages into M2c

subtype that contributes to fibrosis through producing

and secreting TGFβ. Although IL-10 may be also

secreted by macrophages (apparently the case for IL-4

and IL-13 here), the high amount of IL-10 detected from

neutrophils and the relative importance of neutrophils in

ALI inflammation suggest that the effects of neutrophils

are non-negligible here. It may be interesting to test

whether blocking neutrophil-derived IL-10 could

suppress M2c polarization of macrophages and thus

reduce fibrosis post ALI.

To summarize, here we showed novel information on

the precursor cells responsible for exosome-cytokines

after ALI, and demonstrate an interactive and dynamic

crosstalk among major cell types in the inflammatory

response during ALI.

MATERIALS AND METHODS

Animals and protocols

All mouse experiment protocols were approved by the

Animal Research and Care Committee at Tongji

University. All experiments were performed in

accordance with the guidelines from the Animal Research

and Care Committee at Tongji University. Specific

pathogen free (SPF) Balb/c mice (aged 10 weeks, weight

20g) were supplied by Shanghai Model Organisms

Center (Shanghai, China). Mice were anaesthetised

(intraperitoneal ketamine 90 mg/kg; xylazine 10 mg/kg),

and 20 µg ‘ultrapure’ LPS (Sigma-aldrich, St Louis, MO,

USA) in 50 µL was instilled intratracheally (i.t.). After 1

or 4 or 8 or 16 or 32 hours, animals were euthanised and

tracheostomised to obtain BALF specimens.

Permeability index and analysis of BALF-exosomes

For retrieval of BALF, airways were flushed with 0.8

ml PBS. The permeability index was determined in


BALF collected at different time points after ALI.

Permeability index is a quantitative marker for

vascular leakage. BALF samples were recovered,

followed by isolation of BALF-exosomes with total

exosome isolation kit (Catalog number: 4478359;

Invitrogen), which applies sequential centrifugation at

200g, 5 minutes and 4oC to remove cells and large

particles, and at 20000g, 30 minutes and 4oC to isolate

exosomes. Visualization of exosomes was done

by electric microscopy and expression of CD9,

CD63, CD81, ALIX and TSG101 was detected by

RT-qPCR. Purified BALF-exosomes were then

incubated with fluorescence-conjugated antibodies

against CD11c/F4/80 (to identify alveolar macro-

phage-derived exosomes), EpCAM (to identify

epithelial cell-derived exosomes) or CD11b/Ly6G (to

identify neutrophil-derived exosomes), and then

analysed by flow cytometry. The absolute counts for

corresponding exosomes were assessed using

counting beads.

Quantitative RT-PCR

Total RNA was extracted by RNeasy kit (Qiagen,

Valencia, CA, USA). Total RNA is transcribed by

Omniscript reverse transcription kit (Qiagen) to

generate complementary DNA (cDNA), which was

used as templates in RT-qPCR. The primers used for

RT-qPCR were also purchased from Qiagen. A 2-∆∆Ct

method was used for quantification of the relative

mRNA levels, and the values of gene expression were

obtained by sequential normalization of the values to

GAPDH and the experimental controls.

ELISA

Total Protein was extracted using RIPA buffer (Sigma-

Aldrich). Protein concentration was determined using a

BCA kit. ELISA for mouse TNFα, IL-1β, IL-6, IL-4,

IL-10, IL-13, TGFβ and FGF used ELISA kits (R&D

Systems, Los Angeles, CA, USA), according to the

manufacturer’s instruction.

Statistical analysis

All values represent the mean ± standard deviation

(SD). Statistical analysis was carried out using a two-

way analysis of variance (ANOVA) test followed by the

Fisher’s Exact Test to compare two groups using

GraphPad Prism 7 (GraphPad, Chicago, IL, USA) soft-

ware. A value of p<0.05 was considered statistically

significant after Bonferroni correction.

CONFLICTS OF INTEREST

The authors have declared no conflicts of interest.

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